The overall goal of this proposal is to provide the principal investigator (PI) with the experience and skills necessary to become an independent investigator in the field of glaucoma research. The PI's Doctoral and Postdoctoral research were in the field of glaucoma. In his Doctoral research he developed in vivo quantitative confocal imaging approaches to study progressive neural damage. His postdoctoral research was in trabecular meshwork and aqueous humor cell biology and physiology with respect to glaucoma. His specific cell biological focus was in cytoskeleton and cell- extracellular matrix interactions, with correlation to functional changes in live monkey hydrodynamic studies. He now proposes to build on his training background in imaging and trabecular meshwork biology within the research field of glaucoma. He seeks to develop new skills and experimental approaches to study a putative regulatory mechanism of aqueous humor outflow. Elevated IOP is the major risk factor for glaucoma but what goes wrong in the disease process to cause IOP elevation is unknown. The broad long-term goal of this proposal is to understand aqueous humor outflow regulation. The present scientific focus is to seek to better understand a putative regulatory mechanism for intraocular pressure (IOP). The proposal's hypothesis is that contractility of the trabecular meshwork (TM) modulates the outflow resistance of the tissue. The following aims are proposed to address the hypothesis in the live mouse: Aim 1: Establish and test assays for TM contractility and outflow resistance; Aim 2: Study TM contractile function and outflow resistance in a suitable animal model. For Aim 1, an assay for outflow resistance using perfusion techniques will be established. Next a contractility assay involving histomorphometry, immunohistochemistry and Western blotting will be assembled. These assays will be used to evaluate the TM's contractile tone after exposure to lysophosphatidic acid (LPA) and transforming growth factor-22 (TGF22), agents that enhance TM contractility. The RGS2 homozygous knockout (RGS2-/-) mouse has a contractile vascular phenotype and hypertension due to altered G-protein signaling. This impairment also causes the TM to become more contractile. That the mouse develops a lower IOP than normal suggests that the increased contractile tone decreases outflow resistance. For Aim 2, IOP, outflow resistance and contractility assays will be performed in RGS2-/- and wild- type mice. To alter contractility further and putatively drive it to a more heightened state, Caldesmon siRNA will be delivered to the TM via the anterior chamber. After siRNA validation studies to confirm silencing, IOP, contractility and outflow resistance assays will be performed. This stepwise approach potentially provides insights into tissue and molecular regulatory mechanisms affecting the TM's outflow resistance. siRNA, if successfully delivered in the mouse in vivo, will provide a rational basis for answering future questions of both mechanistic and therapeutic nature. This investigation will be based at the Department of Ophthalmology of the University of Southern California. The Department of Ophthalmology here has a strong tradition of fostering basic vision research and clinical scientists. The proposed research will be conducted in dedicated space within the Doheny Vision Research Center, which also houses the Institute's Core facilities that will support the PI's research. The PI will have considerable protected time for research and a plan of didactic education. The PI's research and career development will proceed under the mentorship of Sarah Hamm- Alvarez, PhD, a cell and molecular biologist with expertise in cytoskeleton interactions, related mouse biology, cellular imaging, and pharmacology and drug delivery. The PI will have as a co-mentor Mark Humayun, MD PhD, a clinician scientist and bioengineer with expertise in developing microelectronic systems for the eye and biophysical analysis, which is pertinent to studying the sub-microfluidics of the mouse aqueous outflow system. Paul Kaufman, MD, an expert in live animal aqueous physiology, the outflow pathways of the eye and glaucoma therapies, will provide collaborative support. The proposal addresses a research question of relevance to glaucoma. In the course of the research, didactic activities, and mentorship within a supportive environment, the PI will gain invaluable knowhow and skills for developing a career as an independent researcher and clinical scientist. PUBLIC HEALTH RELEVANCE: Project narrative Glaucoma, the leading cause of irreversible blindness worldwide, has as its major risk factor elevated intraocular pressure. This project studies a potential mechanism for regulating intraocular pressure that can help us better understand glaucoma and ways to treat it.